Reagents for the diagnosis of infectious mononucleosis and preparation of same

ABSTRACT

SHEEP OR HORSE BLOOD IS SPECIALLY TREATED TO PRODUCE AN ANTIGEN WHICH IS READILY AGGLUTINABLE BY HETEROPHIL TYPE ANTIBODIES PRESENT IN THE PATIENTS SCRUM. THE ANTIGEN POSSESSES PROPERTIES OF STABILITY, SENSITIVITY AND AGGLUTINABILITY WHICH ARE UTILIZED IN A SEROLOGICAL DIAGNOSTIC TEST FOR INFECTIOUS MONONUCLEOSIS. TWO OTHER ANTIGENS, PREPARED FROM BEEF BLOOD AND FROM GUINEA PIG TISSUES, ARE USED IN THE TEST TO DIFFERENTIATE THE INFECTIOUS MONONUCLEOSIS TYPE ANTIBODY FROM THE FORSSMAN TYPE.

United States Patent 3,826,821 REAGENTS FOR THE DIAGNOSIS OF INFECTIOUSMONONUCLEOSIS AND PREPARATION OF SAME Joseph Zichis, Chicago, 11].,assignor to Beckman Instruments, Inc.

No Drawing. Continuation-impart of abandoned application Ser. No.583,433, Sept. 30, 1966. This application Feb. 2, 1971, Ser. No. 112,062

Int. Cl. G01n 31/00, 31/02, 33/16 US. Cl. 424-12 Claims ABSTRACT OF THEDISCLOSURE This application is a continuation-in-part of applicationSer. No. 583,433, filed Sept. 30, 1966, now abandoned.

DESCRIPTION OF THE INVENTION This invention relates to the provision ofa diagnostic test for infectious mononucleosis, and more particularly,to a process for preparing reagents for use in the test and method ofperforming the test.

Infectious mononucleosis is a disease of unknown etiology. It haspreviously been discovered that in the course of this illness thepatient develops a heterophil antibody in his serum which is differentfrom the Forssman type heterophil antibodies. Since the latter are knownto occur in many normal individuals, and in various disease conditions,and because the heterophil antibodies of infectious mononucleosis andForssman type antibodies possess certain common characteristics (e.g.they both react with antigens not associated with their production andboth types agglutinate sheep and horse erythrocytes it has beenrecognized that laboratory diagnostic tests for infectious mononucleosismust be capable of distinguishing between the two.

One widely used test (known as the Davidsohn test employs raw, washedsheep erythrocytes in a presumptive test and differentiates theheterophil antibodies by absorption with suspensions of guinea pigkidneys and beef erythrocytes. The test is performed by a tube method.It involves comparative titrations of the absorbed and unabsorbed testserum. The final results are recorded after two hours of incubation.Other methods which have been used employ treated sheep or horseerythrocytes and are performed by the slide technique.

A primary object of the present invention is to provide a technique fordiagnosing infectious mononucleosis which includes the preparation andutilization of unique antigens which function to accurately detect andidentify the infectious mononucleosis heterophil antibody. Anotherobject is to provide a diagnostic technique in which presumptive anddifferential tests for this heterophil antibody are combined into asingle test making it possible to estab- Paul .I. R. and Bunnell, \V.W., Am. J'. Med. Sci. 183 90, gg2igijley, G. H. and Raffel, S., J. Clin.Investigation, 14:

o. Barrett, A. M., J. Hyg., 41: 330, 1941, Hoff, G. and Bauer, 8.,J.A.M.A., 194 351, 1965.

Davidsohn, 1., et al., Am. J. Clin. Path., 21:1101, 1951, Davidsohn, I.,J.A.M.A., 108 289, 1937.

3,826,821 Patented July 30, 1974 lish a diagnosis of infectiousmononucleosis within just a few minutes time.

Yet another object of this invention is to provide novel diagnosticprocedures and reagents which can be routinely employed without the needof elaborate and costly laboratory facilities, and which can provideaccurate results in substantially faster time than previously wasthought possible.

In carrying out the invention heterophil type antibodies in the patentsserum are detected by reacting the serum with an antigen speciallyprepared from sheep or horse blood, and the infectious mononucleosisantibody in the patients serum is identified by means of differentialneutralization reactions of the serum with antigens specially preparedfrom beef blood and from guinea pig tissues, all of which reactions maybe readily visually observed (for example by using the glass slidetechnique). Thus, the invention contemplates detection of the presenceof heterophil antibodies in the patents serum through reaction with aspecial antigen prepared from sheep or horse blood, and differentiationof the infectious mononucleosis type heterophil antibody from theForssman type through use of neutralizing antigens specially preparedfrom beef blood and from guinea pig tissues.

The preparation and standardization of the various special antigens usedin the test may be carried out as described below.

SPECIAL ANTIGENS FROM SHEEP -OR HORSE BLOOD AND FROM BEEF BLOOD ice Thespecial antigens from sheep or horse blood and from beef blood areprepared by treating citrated sheep, horse, or beef blood with anaqueous solution containing borate ions preferably along with a suitableanti-contaminant which prevents the growth of microorganisms that mayaffect the antigen and in this sense acts as a preservative, such assodium azide, and a suitable agent for enhancing the isotonic characterof the solution (e.g. sodium chloride). The solution should be isotonicand neutral, i.e. have a pH close to 7. After treatment the mixture isincubated and the sediment, containing the antigen, is separated out.

Various combinations of acids and salts capable of producing borate ionsin solution may be used. Thus combinations of a borate salt of an alkalimetal, e.g. sodium borate, with an acid such as ascorbic or isoascorbic,acetic or hydrochloric acid have been used successfully. Oneparticularly useful combination is boric acid and sodium hydroxide.

One representative way in which the special antigen from sheep or horseblood may be prepared is as follows:

1. 500 cc. of sheep or horse blood is added to 600 cc. of a watersolution containing 3.8% by weight of sodium citrate.

2. To 2000 cc. of distilled water add 18 gms. of CF. sodium chloride, 44gms. of sodium borate, 35 gms. of isoascorbic acid, and 5.0 gms. ofsodium azide. Then if necessary adjust to pH 7.0, using either sodiumborate or isoascorbic acid.

3. Combine the citrated blood of (1) with the mixture of (2) and mixthoroughly.

4. Incubate the mixture at about 37 C. with stirring at intervals .(e.g.three times a day). Allow settling to take place overnight. Continueuntil such settling indicates the formation of a White-grayish layer(the antigen). This usually occurs within five to eight days. Allow theantigen formation to continue for about three additional days. The 37 C.temperature is an optimum in the sense that the antigen tends to bedestroyed at higher temperaures, while lower temperatures adverselyextend the reaction time beyond the preferred time period stated above.

5. After incubation as described in step (4), stir the mixture andcentrifuge for 30 minutes at 4500 r.p.m. to separate the antigen fromthe remaining materials such as the hemoglobin, plasma, cell proteinsand sodium citrate. Centrifugation produces three layers, a bottom layerof heavy cellular material, a middle layer of the white-grayish materialwhich contains the antigen, and a top layer of supernatant liquid.

6. Discard both the supernatant liquid (top layer) and the heavycellular material (bottom layer). Wash the middle layer, containing theantigen, with saline solution by centrifugation, once more discardingtop and bottom layers and retaining the middle antigen containing layer.Repeat the washing until the supernatant is clear (indicating that thehemoglobin and other separated materials (see step 5 above) have beenremoved). Usually three washings suffice.

7. The white-grayish sediment constitutes the special sheep or horseblood antigen. The antigen is taken up in 500 cc. of saline solution,and preserved by adding 0.2% by weight sodium azide.

Where it is desired to use the sodium hydroxide and boric acidcombination mentioned previously, step (2) above is replaced by thefollowing, all other steps remaining the same: To 3000 cc. of distilledwater heated to 37 C. add 27.5 guns. of CF. sodium chloride, 90 gms. ofboric acid, 120 cc. of 1 N NaOH, and 7.0 gms. of sodium azide. Adjust topH 7.0 if necessary.

The antigen from beef blood is prepared following the same proceduresdescribed above except that beef blood is used rather than sheep blood.

The resulting sheep or horse blood antigen may be standardized asfollows:

1. Make serial dilutions of the antigen in saline solution from one partantigen to two parts saline solution to one part antigen to thirty-two(32) parts saline solution.

2. Place a drop of each dilution on a separate one inch square marked ona glass plate.

3. To each. such drop, add a drop of standard positive infectiousmononucleosis serum.

4. Mix the reagents on each square with a separate wooden applicator.

5. Manipulate the glass plate over an indirect light and observe theagglutination reactions.

The dilution that reacts the fastest and produces the heaviestagglutination constitutes the titer of the antigen. The final antigenthen is made up to this dilution.

It is usually found that the antigen prepared from horse blood issomewhat more sensitive than that prepared from sheep blood.

The beef antigen may be standardized by demonstrating its neutralizingeffect against a standard positive infectious mononucleosis serum.

SPECIAL ANTIGEN FROM GUINEA PIG TISSUES The special antigen from guineapig tissues is prepared by making a saline extract of guinea pig kidney,spleens and lungs. One technique which may be used is as follows:

1. Grind the guinea pig tissues in an Osterizer or like blender at fullspeed for five minutes, and make an 18% by weight suspension of tissuesin saline solution.

2. Add about 0.2% by weight sodium azide.

3. Store at about 2-5 C. for approximately 24 hours.

4. Centrifuge at 2000 rpm. for 15 minutes.

5. Decant and save the supernatant liquid; discard the sediment.

6. Heat the supernatant liquid for 20 minutes at 56 C.

7. Centrifuge again at 2000 rpm. for 20 minutes.

8. Decant and save the supernatant liquid which contains the antigen;discard the sediment. If the supernatant liquid is found not to be ofsufficient titer, when standardized as described below, it should beconcentrated to the A serum collected from a proven case of the disease,and having at 1-160 titer as determined by the Dav dsohn Test.

4 I desired potency using a suitable evaporation technique. If thesupernatant liquid is too concentrated it should be diluted.

The guinea pig antigen may be standardized by testing serial dilutionsof it with a Forssman heterophil antiserum or a positive infectiousmononucleosis serum as described above. The Forssman antibodies areneutralized (thus agglutination does not occur), and the infectiousmononucleosis antibody is not.

TEST

The test which results from practice of the invention makes use of thediscovery that the specially prepared antigens described above eachperform different functions leading to specific detection andidentification of the heterophil antibody of infectious mononucleosis.Thus, the specially prepared sheep or horse blood antigenagglutinatively reacts with, and thus detects the presence of, anyheterophil type antibody, including both the Forssman and the infectiousmononucleosis types. The other two specially prepared antigens thenaccurately distinguish between the Forssman and the infectiousmononucleosis type heterophil antibodies. The specially prepared beefblood antigen functions to inactivate or neutralize the infectiousmononucleosis type heterophil antibody but does not neutralize theForssman type. The specially prepared guinea pig antigen, on the otherhand, neutralizes the Forssman type but not the infectious mononucleosisantibody. The manner in which this discovery is utilized will be seenfrom the below described test procedure.

To perform the test, one drop of the patients serum is placed in each ofthree one inch squares on a marked glass plate.

The separately prepared and standardized sheep or horse blood, beefblood and guinea pig tissue antigens are individually thoroughly shaken.Then to the first square on the glass plate a drop of the beef bloodantigen is added; to the second square a drop of the guinea pig tissueantigen is added; and to the third square a drop of saline solution isadded. To a fourth square two drops of saline solution are added.

Then the reagents in each of the first three squares are mixed withseparate wooden applicators and allowed to stand for about one minute.

Next a drop of the special sheep or horse blood antigen is added to eachof the four squares. Again the reagents in each square are mixed withthe wooden applicators as before.

Finally the glass plate is manipulated gently over an indirect light fornot more than about two minutes and the presence or absence ofagglutination reactions is visually observed. The final agglutinationreading should be made five minutes later.

In a'positive test, the heterophil antibody of infectious mononucleosispresent in the patients serum is neutralized or inactivated by the beefblood antigen (in the first square), but not by the guinea pig tissueantigen (in the second square). Thus, when the sheep or horse bloodantigen is added to each of the glass squares agglutination does notoccur in the presence'of the beef blood antigen (first square), but doesoccur in the presence of the guinea pig tissue antigen (second square).In addition, agglutination also occurs in the third square where thesheep or horse blood antigen reacts with the patients serum withoutother antigens being present. The mixture in the fourth square serves tocontrol the granulation that may be present in the sheep or horse bloodantigen.

A negative test is manifested in either of two ways, viz there will beno agglutination of the serum with any of the three antigens (in whichcase no heterophil antibodies are present), or there will beagglutination of the serum with the sheep or horse blood antigen (thirdsquare) and in the presence of the beef blood antigen (first square) butnot in the presence of the guinea pig tissue antigen (second square).The latter situation shows the presence of the Forssman type antibodiesbut not of the infectious mononucleosis type.

In rare cases, agglutination may occur in the presence of all threeantigens. Such a reaction may result if the specimen contains both theForssman and the specific infectious mononucleosis antibodies. It mayalso result if only one of the two antibodies is present, but its titeris higher than the beef or guinea pig antigens are capable ofneutralizing. A differential titration of the specimen will resolvethese conditions.

While the types of agglutination that occur in a positive test reactionrange from coarse to fine clumping, any detectable agglutination isinterpreted as a positive reaction. Since with some sheep or horse bloodantigens, a fine granulation of the antigen may occur, the antigensaline control procedure carried out using the fourth glass squareserves to distinguish this granulation from the positive reactions givenin the test.

Occasionally, the test should be performed with a positive infectiousmononucleosis serum, so that the typical and distinct action of theantigens may be observed.

Nineteen hundred (1900) sera from suspected cases of infectiousmononucleosis that gave positive agglutination by the Davidsohnpresumptive test have been tested according to the present inventionutilizing the sheep blood antigen on a comparative basis with theDavidsohn differential method. The presumptive titer of these seraranged from 1-7 to 1-896.

When these sera were subjected to the Davidsohn differential methodeight hundred twenty seven (827) tested positive for infectiousmononucleosis, and ten hundred seventy three (1073) tested negative.When the same sera were tested using the method of the presentinvention, a total of eight hundred thirty three (833) tested positive(including the 827 which were positive according to the Davidsohn test),and ten hundred sixty seven (1067) tested negative.

To confirm the specificity of the method of the present invention, threehundred (300) sera that tested negative according to the Davidsohnpresumptive test were tested using the method of the invention and werealso found to be negative.

Two hundred and fifteen (215) sera from suspected cases of infectiousmononucleosis that tested positively using the sheep blood antigen asdescribed herein, were also tested according to the present inventionutilizing the horse blood antigen. All the sera likewise testedpositive. The comparative results showed that the horse blood antigenwas more sensitive than the sheep blood antigen.

While the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives,modifications, and variations will become apparent to those skilled inthe art in light of the foregoing such alternatives, modifications, andvariations as fall within the spirit and broad scope of the appendedclaims.

I claim as my invention: 1. The method of preparing an antigen for useas a serological reagent in the diagnosis of infectious mononucleosiswhich comprises:

treating sheep erythrocytes with an isotonic aqueous solution having apH of about seven and containing sodium, chloride and borate ions and apreservative;

incubating the resulting mixture of erythrocytes and isotonic solutionat about 37 C. for about five to about eleven days; and

separating the resulting stable serologically active white-grayishantigen from the residual liquid and blood substances. 2. The diagnosticantigen for infectious mononucleosis prepared by the method of Claim 1.

3. The method of preparing an antigen for use as a serological reagentin the diagnosis of infectious mononucleosis which comprises:

treating beef erythrocytes with an isotonic aqueous solution having a pHof about seven and containing sodium, chloride and borate ions and apreservative;

incubating the resulting mixture of erythrocytes and isotonic solutionat about 37 C. for about five to about eleven days; and

separating the resulting stable serologically active white-grayishantigen from the residual liquid and blood substances.

4. The diagnostic antigen for infectious mononucleosis prepared by themethod of Claim 3.

References Cited UNITED STATES PATENTS 5/1969 Richheimer 424-148 2/1969Hoff 424-12 OTHER REFERENCES 50 ALBERT T. MEYERS, Primary Examiner A. l.FAGELSON, Assistant Examiner US. Cl. X.R.

description. Accordingly, it is intended to embrace all 55 4241.3, 95,101, 148

